Effect of food contamination and collection material in the measurement of biomarkers in saliva of horses.

This study aims to evaluate the effect of the presence of food and the material used in a panel of biomarkers in saliva of horses. For the food effect study, clean saliva was incubated with a known amount of food consisting of oats, hay or grass. Significant changes were observed when saliva was incubated with oats for total protein (P = .050) and phosphorus (P = .008), with grass for total protein (P = .037), salivary alpha-amylase (sAA, P = .018), total esterase (TEA, P = .018), butyrilcholinesterase (BChE, P = .037), adenosine deaminase (ADA, P = .037), and total bilirubin (P = .018), and with hay for sAA (P = .018), phosphorus (P = .037), γ-glutamyl transferase (gGT, P = .004), and creatine kinase (CK, P = .016). For the material-based collection study, saliva using a sponge and a cotton role at the same time were collected and compared. Lower values were obtained in clean saliva collected with cotton role compared to sponge for sAA (P = .030), TEA (P = .034), BChE (P = .003), gGT (P = .002) and cortisol (P < .001) In conclusion, the presence of food and the material used for its collection, can influence the results obtained when analytes are measured in saliva of horses.

Identification of possible new salivary biomarkers of stress in sheep using a high-resolution quantitative proteomic technique.

The aim of this study was to identify biological pathways and proteins differentially expressed in the saliva proteome of sheep after the application of a model of stress, using high-resolution quantitative proteomics. In addition, one of the proteins differently expressed was verified and evaluated as a possible biomarker of stress in this species. Saliva paired samples from eight sheep before and after the application of a model of stress based on shearing were analysed using tandem mass tags (TMT). The TMT analysis allowed for the identification of new stress-related metabolic pathways and revealed 13 proteins, never described in saliva of sheep, that were differentially expressed between before and after the stress. Six of these proteins pertain to four major metabolic pathways affected, namely: canonical glycolysis, oxygen transport, neural nucleus development, and regulation of actin cytoskeleton reorganization. The rest of proteins were unmapped original proteins such as acyl-coenzyme-A-binding protein; complement C3; alpha-2-macroglobulin isoform-X1; type-II small proline-rich protein; lactoferrin; secretoglobin family-1D-member; and keratin, type-II cytoskeletal 6. Of these proteins, based on its biological significance and specific immunoassay availability, lactoferrin was selected for further validation. The immunoassay intra- and inter-assay coefficients of variation were lower than 13%. The method showed good linearity under dilution and recovery, and the detection limit was low enough to detect salivary lactoferrin levels. A significant decrease (P < 0.01) in salivary lactoferrin concentration in the sheep following the application of the model of stress was observed, suggesting that this protein could be a potential salivary biomarker of stress situations in sheep.

Biomarkers of oxidative stress in saliva of sheep: Analytical performance and changes after an experimentally induced stress.

Oxidative stress can affect animal's health and the quality of its final products. The oxidative status can be evaluated by the measurement of both oxidant and antioxidant biomarkers. The use of saliva as a sample is preferable to blood, as individuals with limited training can collect it easily and non-invasively with minimal stress to the animal. The aim of this study was to perform an analytical validation of automated assays of the ferric reducing ability of plasma (FRAP), the cupric reducing antioxidant capacity (CUPRAC), the Trolox equivalent antioxidant capacity (TEAC) and uric acid as antioxidant biomarkers and of the advanced oxidation protein products (AOPP) and hydrogen peroxide (H2O2) as oxidant biomarkers in saliva samples of sheep, and to evaluate their possible changes after stress induced by shearing. All assays produced acceptable results in the analytical validation, from which it can be concluded that oxidative stress biomarkers such as FRAP, CUPRAC, TEAC, uric acid and AOPP and H2O2 can be measured in sheep saliva. In addition, acute stress due to shearing could produce an oxidative stress response in sheep and subsequently increase antioxidants in order to protect cells from damage.

Validation of an assay for quantification of alpha-amylase in saliva of sheep.

The objective of this study was to develop a time-resolved immunofluorometric assay (TR-IFMA) for quantification of salivary alpha-amylase in sheep. For that purpose, after the design of the assay, an analytical and a clinical validation were carried out. The analytical validation of the assay showed intra- and inter-assay coefficients of variation (CVs) of 6.1% and 10.57%, respectively and an analytical limit of detection of 0.09 ng/mL. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution. For clinical validation, a model of acute stress testing was conducted to determine whether expected significant changes in alpha-amylase were picked up in the newly developed assay. In that model, 11 sheep were immobilized and confronted with a sheepdog to induce stress. Saliva samples were obtained before stress induction and 15, 30, and 60 min afterwards. Salivary cortisol was measured as a reference of stress level. The results of TR-IFMA showed a significant increase (P < 0.01) in the concentration of alpha-amylase in saliva after stress induction. The assay developed in this study could be used to measure salivary alpha-amylase in the saliva of sheep and this enzyme could be a possible noninvasive biomarker of stress in sheep.

L'objectif de la présente étude était de développer un test immunofluorométrique en temps résolu (TIMF-TR) pour la quantification de l'alpha-amylase salivaire chez le mouton. À cette fin, suite au design du test, une validation analytique et clinique fut effectuée. La validation analytique du test a montré des coefficients de variation (CV) intra- et inter-tests de 6,1 % et 10,57 %, respectivement, et une limite de détection analytique de 0,09 ng/mL. Le test a également montré un haut niveau de précision, tel que déterminé par la linéarité suite aux dilutions. Pour la validation clinique, un modèle de test de stress aigu a été mené afin de déterminer si des changements significatifs attendus de l'alpha-amylase étaient détectés dans le nouveau test développé. Dans ce modèle, 11 moutons étaient immobilisés et confrontés avec un chien de berger afin d'induire le stress. Des échantillons de salive ont été obtenus avant l'induction du stress et 15, 30, et 60 min par la suite. Le cortisol salivaire a été mesuré à titre d'indicateur de référence du stress. Les résultats du TIMF-TR ont montré une augmentation significative (P < 0,01) de la concentration d'alpha-amylase dans la salive après l'induction du stress. Le test développé au cours de cette étude pourrait être utilisé afin de mesurer l'alpha-amylase salivaire dans la salive de mouton et cet enzyme pourrait être un biomarqueur non-invasif du stress chez le mouton.(Traduit par Docteur Serge Messier).